Nextera dna library prep protocol

DNA was extracted from whole-blood samples or muscle samples using the DNeasy Blood & Tissue Kit (QIAGEN), following manufacturer recommendations for maximizing yield and quality. Concentration was assessed by Qubit 3 (Invitrogen) and 50 ng of DNA were used as input for whole-genome sequencing (WGS). Libraries were prepared using the Nextera DNA Library Prep protocol (Illumina catalog #FC-121-1030). Briefly, DNA was added to a 10 μl reaction containing 5 μl of TD buffer and 1 μl of tagment DNA enzyme (TDE1), then incubated at 55°C for 5 minutes. Tagmentation reactions were cleaned using 2x concentration of Ampure XP beads (Beckman Coulter), then 10 μl of cleaned DNA were added to a 24 μl PCR reaction including 1x NEBNext Q5 master mix (New England Biolabs) and 0.42 μM each of indexed P5/P7 primers. Libraries were amplified using six cycles of PCR and cleaned using 0.65x concentration of Ampure XP beads (Beckman Coulter). Libraries were pooled equimolarly and sequenced on either the Illumina HiSeq X or NovaSeq 6000 platforms (2×151 bp sequencing) to a median coverage of 11.54x.